The effects of 10−3 M iodoacetate (IAA) and (or) 10−3 M dinitrophenol (DNP) on Na and K fluxes and contents and on adenine nucleotide levels of isolated rat uterine horns were studied. Early 22Na efflux was slightly increased by DNP in the fresh and Na-rich tissues. IAA and DNP alone or together reduced 22Na efflux from the larger cellular fraction (No. 2) in both fresh and Na-rich tissues. 22Na efflux from the smaller cellular fraction (No. 3) was accelerated by IAA and by DNP in Na-rich tissues. DNP increased 22Na influx in both types of tissue and caused net Na gain and K loss. In fresh tissues IAA or IAA plus DNP accelerated 22Na influx, but slowed this influx in Na-rich tissues. In fresh tissues the ATP content was reduced by 50% by DNP. After a 60-min exposure with IAA and a 15- to 20-min exposure with IAA plus DNP, the ATP levels were negligible. The onsets of action of IAA or of IAA plus DNP on Na fluxes were correlated with ATP depletion, but early acceleration of 22Na efflux by DNP was not. In fresh tissues 42K influx was slightly decreased at the time of ATP depletion and the influx was further slowed as tissue potassium was replaced by sodium. IAA plus DNP increased K efflux in 10 min and IAA alone increased K efflux after 100 min. Thus K flux changes were not well correlated with ATP depletion. Substitution of K for all the sodium in the bathing media did not alter the quality of the effects of IAA or IAA plus DNP on sodium efflux. When prolonged glucose depletion eliminated ATP and ADP, the effects of IAA could not be duplicated. But IAA alone, or with DNP, still caused alterations in the 22Na efflux. Therefore IAA acted on ion fluxes by a mechanism other than ATP depletion. Both fresh and Na-rich tissues swelled after ATP depletion. An effect on internal osmotic pressure rather than ATP-depletion per se was postulated. Other studies showed that Na-rich tissues were resistant to shrinking by hypertonic sucrose and became more so secondarily after ATP depletion because of increased sucrose permeability. Evidence from studies of swelling, as well as flux data, suggested that at least two Na pumps were present. Both were ATP-dependent. One was ouabain-sensitive and exchanged Na for K, while the other was ouabain-insensitive and controlled movement of Na with water.